The present invention relates to a method for producing a cysteine-free peptide which comprises producing a fused protein or polypeptide and subsequently subjecting the thus obtained protein or polypeptide to cleavage of the peptide linkage at the cysteine residue of said fused protein or polypeptide.
In order to produce a peptide using gene recombination technology, the peptide expressed in the form of a fused protein is frequently employed since peptides are susceptible to be cleaved intracellularly. Known methods of cleaving the desired peptide from the fused protein include a chemical method using cyanogen bromide (Itakura et al., Science, 198, 1056 (1977)) or an enzymatic method using enzyme Factor Xa (Nagai et al., Methods in Enzymology, 153, 46 (1987)).
When cyanogen bromide is used, methionine-containing peptides cannot be produced; and when Factor Xa is used, the yield of the desired peptide is very low.
As the cleavage method, it is known that acylcysteine bond is cleaved by 2-nitro-5-thiocyanobenzoic acid (Seikagaku Jikken Koza (Biochemical Experiment), 1, Chemistry of Protein, pp. 247-250, 1976, Tokyo Kagaku Dohjin).
The present inventors investigated efficient cleavage of the desired peptide from the fused protein without using cyanogen bromide or factor Xa, and found that the desired cysteine-free peptide can be efficiently produced by producing a fused protein comprising a protein having cysteine at its N-terminal and a cysteine-free peptide ligated to the N-terminal using a gene manipulation technique or chemical synthesis and then subjecting the fused protein to a reaction for cleaving the peptide linkage.